The alarming increase in overweight and obese individuals in the US is a major healthcare issue since it contributes to higher rates of type 2 diabetes, vascular disease and cardiovascular events. Because dietary saturated fatty acids have been linked to vascular dysfunction and insulin resistance (a prelude to type 2 diabetes), dietary fatty acid manipulation may be an important target for preventing or reversing vascular abnormalities associated with obesity. The focus of this proposal is to address the mechanisms underlying dietary fatty acid-induced vascular dysfunction. We hypothesize two potential mechanisms: high saturated fat diets may produce abnormal membrane lipid composition which 1) affects formation of specialized membrane compartments (lipid rafts) and activation of signaling molecules or 2) promotes unique sphingolipid signaling pathways within vascular tissue. These abnormal membrane effects of saturated fatty acids may be reversed by enriching the diet with monounsaturated (MUFAs) or polyunsaturated (PUFAs) fatty acids. To test our hypothesis, we will use a mouse model of high saturated fat-induced obesity and manipulate dietary fat composition to address the following Specific Aims: Aim 1: To determine whether replacement of a portion of dietary saturated fats with MUFA or n-3 PUFAs restores vascular function to normal by modifying caveolar versus non-caveolar localization of key signaling molecules. Our working hypothesis is that diets enriched in MUFAs and n-3 PUFAs target endothelial nitric oxide synthase (eNOS) and/or cyclooxygenase (Cox) to membrane lipid rafts differently than a high saturated fat diet by altering membrane composition. This shift in localization defines their activation and vascular function. We will measure responses of small gracilis arteries and aorta from mice on diets enriched in saturated fats, MUFA enriched olive oil or n-3 PUFA enriched menhaden oil in conjunction with membrane fatty acid composition and localization of eNOS and Cox within lipid rafts. We will compare the effects of diet on vascular function with localization of signaling pathways using wild type and caveolin-1 (cav-1) deficient mice devoid of vascular caveolae to separate effects on caveolar versus non-caveolar lipid rafts. Aim 2: To determine whether dietary fatty acid saturation affects sphingolipid signaling pathways- specifically sphingosine kinase and sphingosine-1-phosphate (S1P)- to modify vascular function. Our working hypothesis is that dietary fatty acids promote signaling through formation of unique sphingolipid-based proteins that impact vascular function. We will measure responses of small gracilis arteries and aorta from mice on diets enriched in saturated fats, MUFAs or n-3 PUFAs in conjunction with levels of sphingosine kinase and S1P within vascular tissue. We will assess the effects of dietary fatty acids on sphingosine kinase by comparing expression levels and using pharmacological inhibitors and genetically modified mice. High fat feeding of mice is a relevant model for study of vascular complications of diet since mice become obese, develop insulin resistance and abnormal glucose tolerance similar to type 2 diabetics. High fat diets are the greatest cause of obesity in people. These studies are innovative in directly testing the mechanisms involved in effects of dietary fats on vascular function using a mouse model of obesity: abnormal membrane composition and localization of signaling proteins within specific lipid rafts or fatty acid-dependent signaling pathways. The studies are significant because dietary fats are a known therapeutic target but the optimal diet composition for reversing the progression of cardiovascular disease and the mechanisms involved are not clearly defined.